alpha-chymotrypsin and Aspergillosis

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Topics: Amino Acid Sequence; Animals; Antibodies, Fungal; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Chromatography, Affinity; Chymotrypsin; Guinea Pigs; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Kinetics; Male; Middle Aged; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Substrate Specificity; Vero Cells

The enzyme-linked immuno-filtration assay (ELIFA) permits detection of serological precipitating systems preformed by immunoelectro-diffusion on cellulose acetate strips and simultaneous characterization of immunoglobulins G, M, A and E specific for antigens of Aspergillus fumigatus. We selected 36 sera from 9 patients who were followed up regularly and who suffered from aspergilloma (5 cases), allergic bronchopulmonary aspergillosis (2 cases), Aspergillus bronchitis and invasive aspergillosis (one case each). All of them possessed an IgG-reactive band with chymotrypsin activity. Four different IgE bands were found by ELIFA; one of them was common to all the patients who had anti-A. fumigatus IgE (7 cases out of 9). The IgA and IgM antibodies found in 7 cases out of 9 were both recognized by the same antigenic component but these fractions were distinct from those reacting with the IgE.

Topics: Antibodies, Fungal; Antigen-Antibody Reactions; Antigens, Fungal; Aspergillosis; Aspergillus fumigatus; Chymotrypsin; Humans; Immunodiffusion; Immunoenzyme Techniques; Immunoglobulin A; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunoglobulins

14 enzymatic activities (3 dehydrogenases and 11 hydrolases) were systematically investigated in the extracts of 2 strains of Aspergillus fischeri and 14 strains of A. fumigatus (8 isolated from human or animal lesions named 'pathogenic' strains and 6 strains of saprophytic origin). The enzymic composition of A. fumigatus appeared to be very different from one strain to another but no relationships could be established between enzymic differences and origin of the strains. In addition, the immunoelectrophoretic analysis of these antigenic extracts using rabbits immunsera and sera of patients with pulmonary asperigllosis was performed. The type II chymotrypsic activity, corresponding to C arc, was shown more frequently and gave stronger reaction when antigenic extracts from pathogenic strains were allowed to react with sera of patients. Moreover, when sera of patients revealed a same enzymic activity in a saprophytic strain and a pathogenic strain, the number of precipitin lines was significantly larger with the latter one. Accordingly, antigenic extracts used for precipitin tests had to be preferably prepared from pathogenic strains and tested, for value, against sera of patients with pulmonary aspergillosis.

Topics: Alkaline Phosphatase; Animals; Aspergillosis; Aspergillus; Aspergillus fumigatus; Carboxylic Ester Hydrolases; Catalase; Chymotrypsin; Glucosidases; Humans; Hydrolases; Immunoelectrophoresis; Leucyl Aminopeptidase; Malate Dehydrogenase; Oxidoreductases; Peptide Hydrolases; Precipitin Tests; Species Specificity

Topics: Animals; Antigen-Antibody Complex; Antigens; Aspergillosis; Aspergillus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Chymotrypsin; Cross Reactions; Diagnosis, Differential; Humans; Hydrolases; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Malate Dehydrogenase; Oxidoreductases; Rabbits; Species Specificity

Topics: Animals; Antigen-Antibody Reactions; Aspergillosis; Aspergillus; Blood Protein Electrophoresis; Catalase; Chymotrypsin; Glucuronidase; Humans; Immune Sera; Immunoelectrophoresis; Mycoses; Precipitin Tests; Rabbits; Staining and Labeling